1. Introduction
Cloning by nuclear transfer is a classical example of how experimental models designed for basic research have been subsequently adopted by applied research.
In fact, the transfer of a differentiated cell into enucleated oocytes was suggested first by Spemann (Spemann H., Embryonic Development and Induction. Hafner Publishing Company, New York, 1938: 210–211) to see whether the totipotentiality of a nucleus from a differentiated cell became restricted during development. A large number of papers stemmed from that suggestion and culminated with the work of Briggs and King (Briggs and King, PNAS 1952, 38: 455–461) where nuclei taken from the intestinal epithelium transferred into enucleated Xenopus eggs developed into viable genetically identical animals, in the proper word, a clone.
Technical limitations restricted nuclear transfer to the Amphybia for 30 years before a seminal paper by McGrath and Solter (McGrath and Solter, Science 1983, 220: 1300–1302) initiated the modern development in mammalian cloning. This new era in nuclear transplantation was given further impetus by its use for cloning embryos from domestic species when nuclei of blastomeres taken from 16 cell stage sheep embryos were competent to support full development till normal lambs after transfer into enucleated oocytes (Willadsen S., Nature 1986, 320: 63–65). Since this article was published, many embryologists started to focus on nuclear transfer and several private companies set out for the commercial application of embryo cloning in the cattle industry.
2. Description of the Procedure for Nuclear Transfer
As a result of the efforts made in these years, the following procedure for reconstructing an animal embryo by nuclear transfer has been developed.
a. Individuation of a Recipient Cell
Metaphase II oocytes are commonly used as a recipient cytoplast for nuclear transfer, especially when the procedure is carried out on ungulates. However also fertilised one cell zygotes which had had both pronuclei removed can be used in principle as well.
When the former procedure is adopted, the oocytes, which can be maturated in vitro or in vivo and are usually collected after the appearance of the first polar body, are kept in hepes buffered medium containing Cytochalasin B, an inhibitor of microfilaments that confers the oolemma the plasticity necessary for the further manipulation exposed hereinafter.
b. Enucleation of the Recipient Cell
The oocytes so individuated are in fact usually enucleated prior to the transfer of the nucleus from the donor cell.
In the majority of economically important animals, the oocytes ovulates in the metaphase of the second meiotic division with the 2n chromatids arranged in a metaphase spindle underneath the first polar body. The high content of lipids renders the sheep cytoplasm quite dark thus making impossible, in contrast to other species like mouse and rabbit, the localisation of the chromosomes.
For that reason, the enucleation was originally carried out with a bevelled pipette by aspirating blindly a portion of cytoplasm underneath the first polar body. Because quite often the metaphase spindle migrates under the oocyte cortex, specially in aged oocytes, enucleation was successful only in 70–75% of the cases. The introduction of the vital dye Hoechst 33342, which binds reversibly to DNA (Tsunoda et al., J Reprod Fertil 1988, 82: 173–177) allows the precise localisation of the chromosomes after UV exposure and is widely used in nuclear transfer.
c. Identification of a Donor Cell
Fully or partially differentiated cell or also an undifferentiated cell can be used as a donor cell (the so called “karyoplast”: blastomeres from an early embryo or somatic cells). Such a donor cell can be both cultivated in vitro and abstracted ex vivo, provided that it has however a normal content of DNA and is karyotipically normal. More preferably are used cells in G0 or G1 phase as in fact, it is the only cell cycle stage that guarantees a correct ploidy after embryo reconstruction (Campbell et al., Rev of Reprod 1996, 1: 40–46). No development of embryo has ever been obtained using as a donor cell non-living cells.
d. Possible Genetic Modification of the Chromatin of the Donor Cell
The nucleus of the donor cell can be genetically modified prior to the transfer in the recipient cell, in order to obtain transgenic animals. The term “transgenic” cover not only the animal containing at least one gene from another species in their somatic and germ line, but any animal whose germ line is subjected to technical intervention by recombinant DNA technology.
e. Nucleus Transfer from the Donor Cell to the Recipient Cell
The nucleus of such a donor cell is therefore transferred in the recipient cell. Such a transfer can be carried out by two different procedures: i) cell fusion and ii) nuclear injection. According to the former procedure, which is the most commonly adopted for nuclei of big dimension, the donor cell as a whole is then inserted by the same enucleation pipette used for the enucleation of the recipient cell in the relevant perivitelline space. The reconstructed embryo is then placed in a fusion chamber between two platinum wires whose gap is filled with fusion medium (0.3 M mannitol with 0.050 mM CaCl2 and 0.100 mM MgSO4). Cell fusion is induced by one or more electrical pulses of direct current applied perpendicularly to the two fusion partners. The frequency of fusion is proportionally related to the area of contact between cytoplast and karyoplast and it is usually high in the case of blastomeres, but it becomes sensibly lower in the case of small foetal or somatic cells. The electric pulse opens temporary pores in the adjacent membranes and the cytoplasmic communication established between karyoplast and cytoplast starts the fusion process which is usually completed within one hour; meanwhile, an influx of extracellular calcium ions induces the activation of the oocyte (Sun et al., Development 1991, 115: 947–956).
According to the procedure reported on point ii) above, the nuclear transfer is carried out by microinjection of the donor nuclei with a process which is getting more used when small cells have to be transferred. The final outline of the process is however the same as the transfer carried out by cell fusion with minimal modification (Collas P and Barnes F L., Mol Reprod Dev 1994, 38: 264–267; Wakayama et al., Nature 1998, 394: 374).
f. Transfer of the Embryo in a Recipient Animal
The successfully fused couplets or microinjected oocytes are embedded in agar chips and transferred into the oviduct of a temporary recipient animal. The embedding is a necessary procedure that protects the embryos from immuno-competent cells present in the oviductal lumen.
After seven days the oviducts are flushed back and those embryos that developed to blastocysts or morulae are dissected out from the agar and transferred to synchronous recipients for development to term.
3. Technical Problem
Despite the efforts done by many laboratories, the efficiency of cloning in terms of offspring production has been invariably low and unpredictable (Bondioli et al., Theriogenology 1994, 33: 165–174) for several years.
This low efficiency was primarily due to the empirical approach used for nuclear transfer.
As a consequence of the studies made in the last years some of the technical limitations, which such a low efficiency was due to, were evidenced.
In particular basic studies undertaken in the last five years on the understanding of nuclear cytoplasmatic interaction in reconstructed embryos clarified how at least one of the reasons for the poor development of cloned embryos is the cell-cycle combination, indicating also the ideal combination for reconstructing embryos by nuclear transfer (Collas et al., Biol Reprod 1992, 46:492–500; Barnes et al., Mol Reprod Dev 1993, 36: 33–41; Campbell et al., Biol Reprod 1994, 50: 1385–1393; reviewed by Campbell et al., Rev of Reprod 1996, 1: 40–46).
Another, insurmountable, limit of embryo cloning was identified in the limited number of nuclei obtainable from an individual embryo. In this connection however the ability to use cultured cell lines derived from embryos have offered a large number of advantages over the use of cleavage stage embryos.
In this connection the ideal cells for this purpose were firstly identified in the embryonic stem cell (ES), which however, unfortunately, have not been isolated from embryos of large animals (Galli et al., Zygote 1994, 2: 385–389).
The production of the first sheep cloned from cultured cell line derived from embryos overcame this limit (Campbell et al., Nature 1996, 380: 64–66) opening new and important opportunities in both basic and applied research. The possibility to use cultured cells not only represented the ideal solution to the production of a large number of high genetic merit or genetically modified animals, but also the distinct cell cycle phases displayed by cells in culture opens the possibility to work out the ideal cell-cycle combination for nuclear transfer of differentiated cells.
This seminal paper was in fact immediately followed by other two, the report of the first mammal produced by the nuclear transfer of a somatic cell (Wilmut et al., Nature 1997, 385: 810–813) and the first transgenic lambs produced by nuclear transfer of genetically modified cells (Schieke et al., Science 1997, 278: 2130–2133).
After that two subsequent reports referring to somatic cloning in mice and cow respectively confirmed the fact that the animal resulting from such a process is effectively a clone (Wakayama et al., Nature 1998, 394: 369–374; Kato et al., Science 1998, 282: 2095–2099).
Further progress made in the last years made embryo cloning a reliable technology potentially applicable in animal breeding (Heyman Y and Renard JP, Anim Reprod Science 1996, 42: 427–436; Loi et al., Theriogenology 1997, 48: 1–10; Loi et al., Biol of Reprod 1998, 58: 1177–1187).
Reprogramming of the Nucleus of the Donor Cell
From all these reports it can be concluded that at least a small proportion of somatic nuclei can be developed into viable offspring, and that such a development is strictly consequent to the success in the “reprogramming” of the donor nucleus which occur in the oocyte immediately after transfer. What it is still not clear is the mechanism that regulates this reprogramming which is in fact due to unknown factors present in the cytoplast.
Following transplantation into oocytes, somatic nuclei lose in fact part (or all in the case of Dolly: see Campbell et al., Nature 1996, 380: 64–66) the structural components of the chromosomes that maintain their differentiated state and gain the capacity to execute the regulated expression of genes through embryonic and fetal development (Patterton and Wolffe, Dev Biol 1996, 173: 2–13).
Basically, the differentiation process which starts concomitantly with the activation of the embryonic genome (it does occur in sheep at the 5th cell cycle, 8–16 cell transition) is completely reversed after nuclear transfer, and the transferred nucleus behaves like a zygote.
Somatic nuclei transplanted into mature eggs are therefore remodelled and this morphological change is associated with the re-acquisition of pluripotency, and in some cases, totipotency (Gurdon I., J Embryol Exp Morphol 1962, 10: 622–640).
The molecular machinery responsible of such a remodelling (and therefore reprogramming) of the genome (diploid, somatic or whatever genome) transferred into the oocyte is not yet fully clarified. However as of course no specific and efficient molecular mechanism for this purpose could have been developed in the oocyte during the evolution for a differentiated cell nucleus inserted in the oocyte itself, such a mechanism is deemed to be the same which operates on the aploid spermatozoo genome at the time of activation.
Following fertilisation, the sperm nucleus is in fact rapidly remodelled by the egg cytoplasm to assemble the paternal pronucleus. The assembly of the pronucleus requires the molecular chaperone nucleoplasmin (Philpott et al., Cell 1991, 65: 569–578). Nucleoplasmin specifically removes the basic, sperm specific proteins and on the same time deposits histones H2A.X and H2.B onto chromatin (Philpott and Leno, Cell 1992, 69: 759–767). The resulting specialised chromosomal conformation found in the pronucleus is also maintained in nuclei of cleavage stage embryos (Dimitrov et al., J Cell Biol 1994, 126: 591–601).
Similarly, there is experimental evidence that nucleoplasmin plays also a major role in the remodelling of somatic nuclei strictly linked to the acquisition of totipotency of somatic nuclei, which requires the release of chromatin components and the uptake of structural and regulatory proteins from the cytoplasm (Philpott et al., Cell 1991 65: 569–578; Wangh et al., J Cell Science 1995, 108: 2187–2196).
In particular it has been observed that the specific dissociation of somatic linker histones H1 and H1° associated with the incorporation of oocyte-specific linker histone B4 into the remodelled chromatin mediated by nucleoplasmin, increases the trascriptional competence, and thus the totipotence of somatic nuclei (Dimitrov and Wolffe, the EMBO Journal 1996, 15: 5897–5906).
While however the nucleosomal transition during remodelling have been described in detail (for review see Patterton and Wolffe, Dev Biol 1996, 173: 2–13), the regulation of long-range chromatin structure during development is far less clear. There is increasing evidence that high order chromatin structures play a role in the acquisition and maintenance of the committed status of the cells. In particular, it has been shown that the protein of the SMC (Segregation of Mitotic Chromosome) and chromodomain families are important for transcriptional control (Chang et al., Cell 1994, 79: 459–474; for review see: Patterton and Wolffe, Dev Biol 1996, 173: 2–13).
From the above consideration it was suggested that changes in the chromatin structures can facilitate the reprogramming of the transferred nucleus upon the relevant transfer (Wilmuth I. et al., Nature 1997, vol. 385, pag. 810–813; Campbell et al., 1996), and that more accessible is the chromatin to cytoplasmatic remodelling factors, better chances the nucleus has to be completely reprogrammed upon nuclear transfer.
In particular it was suggested that the reduced transcriptional activity of quiescent cells may be beneficial for reprogramming (Campbell et al., 1996), although no direct comparison has been done with nuclei in different stage of the cell cycle.
Also, the displacement of sequence-specific transcription factors from mitotic chromatin (Martinez-Balbas et al., Cell 1995, 83: 229–238) positively influences the remodelling of mitotic cells into metaphase cytoplasm (Fulka et al., BioEssay 1996, 18: 835–840) and consequently induces a better reprogramming as indicated in mice experiment (Kwon and Kono PNAS 1996, 93: 13010–13013).
Such a displacement is a “physiological” consequence of the reduced metabolic activity in starved G0 cells (Campbell et al., Nature, 1996), and a consequence of a prolonged chromosome condensation in the method suggested by Wakayama (Wakayama et al., 1998). In both cases, the displacement or the termination of transcriptional activity falls into the normal activity of the cell and it is not “per se” responsible for genome reprogramming as clearly indicated by the fact that the phenotype of the cell is stably maintained after both cell cycle stages. However, despite the use of a highly defined synchronous population of G0 nuclei donors, or the uniform, prolonged exposure of the transferred nuclei into the cytoplasmic environment, less than 2% of nuclei are fully reprogrammed and develop into viable young upon nuclear transfer. Considering that the oocyte cytoplasm would normally encounter a transcriptionally inactive sperm nucleus rather than a fully differentiated nucleus, the low efficiency resulting from the above procedures is unsurprising. No alternative procedures have been suggested so far and foremost more invasive procedures that may lead to the loss of cell viability for two reasons: firstly, nuclear transfer is still accomplished by electro-mediated cell transfer, this method working only with living cells, secondly, a no-living cell is commonly believed to not be a good candidate for nuclear transfer.
In spite of the fact that numerous studies have indicated that high temperatures denature proteins and nucleic acids with a melting transition occurring when the temperature exceeds 55° C. (Pain RH., Symp Soc Exp Biol 1987, 41: 21–33), and that the denatured state of proteins is the primary target for degradative enzymes (McLendon and Radany, J Biol Chem 1978, 253: 6335–6337)— and therefore in the specific case also for the specific proteasome activity present in mature oocytes (Saro CK and Hoschi M, J Biochem 1997, 122: 286–293)— the elimination of such factors by the denaturation and subsequent degradation by degradative enzymes, was not considered in art.
In this connection, however it shall be kept into account the proved existence of a real epigenetic “cell memory” which is necessary for maintaining a stable pattern of gene expression in dividing cells, and allows the cellular phenotypes of differentiated cells to be stably propagated through cell division.
DNA methylation and the propagation of specific chromatin structures are in particular suggested as good candidates for the maintenance of the cell memory (Patterson and Wolffe, Dev. Biol 1996, 173: 2–13). Alternatively or concomitantly, some factors might remain bound to mitotic chromosomes acting as bookmark for those genes that must be re-expressed.
The same consideration can be drawn for quiescent G0 cells. These cells are still metabolically active, although at reduced levels, and no longer proliferated unless called to do so by appropriated extracellular signals. Of course, the phenotype of quiescent cells does not change after re-activation indicating that a stable pattern of gene expression is maintained by specific chromatin structures during G0 too.
Accordingly both mitotic and quiescent cells retain the epigenetic cell memory that maintains the differentiated status. The essential condition for a full reprogramming of a somatic cell is therefore its complete remodelling which involves a transition in chromosomal structures and composition associated with the acquisition to carry out the rapid cleavage cycle of early development and to execute the regulated expression of genes through embryonic and fetal development till the birth of a normal, viable animal.
Two distinct approach have been suggested for the induction of full reprogramming of somatic nuclei. The first, postulated by Campbell and co-workers (Campbell et al., Nature 1996, 380: 64–66) says that nuclear quiescence inducted by serum deprivation is the fundamental condition for nuclear reprogramming; the second one, claimed by Wakayama (Wakayama et al., Nature 1998, 394: 369–374), but also anticipated by Campbell (Campbell et al., Nature 1996, 380: 64–66), says that a prolonged exposition of the nuclei into the cytoplasm environment increases the chances for nuclear reprogramming.
However, the importance of nuclear quiescence for somatic nuclear transfer is still controversial. In fact, in the first report on the use of G0 cells as nuclear donors (Campbell et al., Nature 1996, 380: 64–66) no comparison is done with cells in other stages of the cell cycle. The situation did not change in the following report (Wilmut et al., Nature 1997, 385: 810–813) where quiescent cells from three different cell lines, embryo, fetal and adult derived cells have been used as nuclei donors.
Moreover, actively proliferating fetal fibroblast cells have been shown to direct normal embryonic and fetal development in the cow (Cibelli et al., Science 1998. 280: 1256–1258) and no differences in blastocysts production were found between proliferating and quiescent somatic and fetal bovine cells in a recent comparative study (Le Bourhis D et al., Clevage et Insemination 1998, Octobre, n 287, 3–9).
So the question is: why, although nearly all the cells used for nuclei transfer are in G0, only a small proportion, 2%, develop into viable offspring? What helps those cell to gain a full totipotency after nuclear transfer?